Abstract:Intellectualdisability(ID)affects1-3%ofthepopulation,resultingincognitiveandadaptivebehavioral deficits.ManygenesareassociatedwithID,includingmultiplemutationsinKDM5C,anX-linkedgenewhichwe discoveredtobeahistonedemethylaseadecadeago.Inthepastfundingcycle,wefoundthatKDM5Cpatient mutations reduce both protein stability and catalytic activity. We showed that a mouse Kdm5c knock-out (KO) model recapitulated the cognitive and behavioral deficits seen in human patients. Kdm5c bound primarily at promoters in terminally differentiated mouse neurons to modulate methylation at lysine 4 of histone 3 (H3K4), andKdm5clossaffectedtheexpressionofneuronalgenesintheamygdala.Furtherstudiesofconditionalmouse Kdm5cKOshavesuggestedthatKdm5cpotentiallyplaysaneurodevelopmentalrole.Tostudythefunctionof KDM5Cduringneurodevelopmentinahumanmodel,wehavegeneratedpatient-derivediPScelllinesbearing KDM5C mutations, and isogenic lines with the mutations corrected, both of which can undergo neuronal differentiationinculture.ThesecelllinesprovideanunprecedentedopportunitytoexploretheeffectsofKDM5C inawell-definedandexperimentallyaccessiblehumandevelopmentalsystem. The goals of this work are to obtain a comprehensive molecular and cellular understanding of how KDM5C regulateshumanneurodevelopment.Wewillconducthigh-resolutiontime-courseanalysestodetermineexactly which stages of neuronal differentiation, and which cell types, are compromised by KDM5C mutation. We will investigatethefunctionalityofKDM5C-mutantneuronsbyinterrogatingtheexpressionofsynapticmarkersand electrophysiology.Becausesomeaspectsofbraindevelopment(e.g.formationofmultiplecelltypesandtheir organization)arenotrecapitulatedina2Dculturesystem,wewilluse3Dhumanbrainorganoidsgeneratedfrom mutant and corrected iPS cells to investigate the roles of KDM5C in promoting brain growth, generating the appropriate diversity of neural cell types, and facilitating neuronal network connectivity. Our findings will be validatedinvivoduringembryogenesisofWTandKdm5cKOmice,andthecriticaltimingandlocationofKdm5c activitydeterminedbyexpressingordeletingKdm5cinspecificstages/celltypes.Toinvestigatethemolecular mechanisms of the neuronal differentiation defects in KDM5C mutant cells, we will determine transcriptional profiles of mutant and corrected iPS cells during differentiation in 2D and 3D cultures. Because the critical genomic targets of KDM5C (e.g. promoters, enhancers) during neurodevelopment are unknown, we will map KDM5C binding sites genome-wide during 2D neuronal differentiation, and determine how KDM5C mutations alter the chromatin landscape. Finally, we will use biochemical approaches to identifyKDM5C regulators, and investigatetheireffectsonKDM5Cfunctionandneurodevelopment.Thesestudieswillshedcriticalnewlighton KDM5Cfunctionduringneurodevelopment,andprovideabasisfordesigningtherapeuticstrategiestotreatID.